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United States Department of Agriculture

Agricultural Research Service

2011 Annual Report

1a.Objectives (from AD-416)
The long-term goal of this research team is to develop efficient methods of preserving poultry, swine and fish germplasm. Over the next five years we will (1) identify the physiological and biochemical impacts of hypothermic storage on poultry, swine and fish sperm, (2) elucidate the cellular and molecular mechanisms controlling sperm selection, transport and storage in the female reproductive tract of poultry, (3) determine the impact of genetics on the success of semen storage methodology for poultry and swine, and (4) investigate alternative strategies for conserving valuable poultry and swine germplasm. Alternative strategies to be investigated include:.
1)creation of transient pores and/or use of endogenous plasma membrane transporters to deliver antioxidants, cryoprotectants and/or nutrients intracellularly;.
2)development of diets to modify the plasma membranes of sperm from congenic and/or inbred poultry lines to improve cryosurvival; and.
3)development of methods to isolate, propagate, freeze/thaw and transfer poultry spermatogonia to recipient sterilized testes.

1b.Approach (from AD-416)
In the food animal industries, production of offspring that possess economically important traits is most effectively accomplished by artificial insemination (AI) or in vitro fertilization (IVF), where semen from a few males is distributed among a large number of females. The poultry and swine industries use AI in their breeding programs to accelerate genetic advancement, while the striped bass industry relies on IVF. Because of gaps in our fundamental knowledge of sperm biology, the fate of sperm in the oviduct and impact of freezing on sperm function, there has been limited success in the long-term preservation of poultry, swine and bass germplasm, and existing methodologies are not adequate for the needs of these industries. Development of effective semen storage methodology necessitates a scientific foundation addressing the cellular and molecular biology of both the sperm cell and the female cells that interact with sperm after insemination. Experiments in this project will address these fundamental questions by focusing on (1) sperm membrane composition and energetics before and after hypothermic storage, (2) impact of sperm on oviductal epithelial cell gene expression and secretory activity, and (3) potential genetic basis of sperm cryosurvival. Included in this project are several alternative strategies for germplasm preservation: introduction of cryoprotectants intracellularly; dietary modification of sperm cell membranes; and use of cryopreserved testicular cells as an alternative means of male germplasm cryopreservation. This systematic approach will address the gaps in our knowledge and permit development of novel and/or more efficient methods of preserving poultry, swine and fish semen.

3.Progress Report
Research efforts during the past year have impacted the ability to freeze, store and utilize germplasm. With respect to identifying the physiological and biochemical impacts of hypothermic storage on poultry and fish sperm (Objective 1), we have shown that poultry sperm respond favorably to the presence of the monosaccharide sialic acid in semen extender where it improves the number of viable sperm 2-fold after only 2 hours of incubation, and the levels of this carbohydrate were maintained in the sperm plasma membrane. For striped bass sperm, we demonstrated that a modified buffered-saline extender was able to maintain viability, motility, and ATP content at acceptable levels for 48 hours, which provides adequate semen storage prior to in vitro fertilization procedures necessary for hybrid striped bass production. With respect to elucidating the cellular and molecular mechanisms controlling sperm selection, transport and storage in the female reproductive tract of poultry (Objective 2), a second trial has been initiated that employs a micro-dissection technique to isolate individual cells composing the sperm storage sites rather than analyzing heterogenous populations of oviductal cells. With respect to determining the impact of genetics on the success of semen storage methodology for poultry and swine (Objective 3), significant progress was made on the genetic analysis of the freezability of boar sperm. We have shown that sperm freezability is an ideal candidate for marker assisted selection or selection for favorable alleles. Finally, with respect to investigating alternative strategies for conserving valuable poultry and swine germplasm (Objective 4), we found that boar sperm disintegrated when they were treated with a genetically altered Staphylococcus aureus pore-forming protein that we intended to use for loading sperm cells with cryoprotectants, such as trehalose. We will use a different method to synthesize this protein for testing its ability to form pores in sperm. We found that extracellular trehalose treatment was not a good replacement for standard monosaccharides and disaccharides in extenders as it did not improve the cooling and freezing of boar sperm. For poultry sperm, we demonstrated that diet modification improves the fertilizing ability of frozen/thawed turkey sperm, where fertile embryos were obtained up to 12 weeks and hatched poults were obtained up to 5 weeks after a single insemination. This degree of success has not been demonstrated previously for turkey semen.

1. Genetic analysis of boars for better sperm survival after cryopreservation. Discovery of genetic markers for boars with superior sperm cryosurvival would allow boar semen distributers to predict which boars will produce litters from frozen/thawed semen. ARS researchers at Clay Center, NE and Beltsville, MD estimated genetic parameters from 920 ejaculates collected over seven years from 254 boars representing a four breed composite that were frozen and then thawed for the determination of sperm viability and motility. The likelihood of a genetic cause for motion parameters was high for the percentages of motile and viable sperm in both fresh and frozen/thawed sperm samples. Sperm freezability is an ideal candidate for marker assisted selection or selection for favorable alleles and freezability could be improved through genetic selection without negative impact on growth and backfat thickness. This important work was presented at the 7th International Conference on Boar Semen Preservation in August 2011.

2. The surface carbohydrates of rooster sperm are altered during cryopreservation. The carbohydrate-rich zone on the surface of the sperm is essential for immune protection in the female tract and early gamete interactions. An ARS-Beltsville scientist and Research Associate evaluated the effect of 3 different sperm freezing protocols on the sugar components of the sperm membrane. Several key carbohydrates were lost during the freeze/thaw process, indicating that the interaction of proteins and/or lipids to which the carbohydrates were attached (or glycoconjugates) was most likely altered and could be responsible for the poor fertility associated with frozen/thawed sperm. The type of freezing method also affected the degree of carbohydrate damage. Although the glycoconjugates have not yet been identified, it is likely that these cryopreservation-induced changes contribute to the reduced fertility of frozen-thawed chicken semen. This work was published in the Journal of Poultry Science in 2011.

3. Species-specific differences exist with respect to sperm cryopreservation. ARS scientists in Beltsville, MD and Fayetteville, AR evaluated the cryosurvival of turkey and crane sperm frozen in a dimethylacetamide (DMA) cryodiluent supplemented with osmoprotectants and/or ATP. For semen frozen only with DMA, post-thaw sperm motility was greatest with lower DMA concentrations, regardless of species. Turkey sperm frozen with a combination had better post-thaw motility compared to DMA alone; whereas the sucrose/trehalose/DMA combination improved crane sperm viability only with lower concentrations of DMA. The post-thaw motility and viability of crane sperm was improved with a combination of DMA and ATP. These data affirm that there are avian-specific differences in sperm survival after cryopreservation and suggest that post-thaw survival can be enhanced by including species-based osmoprotectant/ATP combinations in a cryodiluent where DMA is the cryoprotectant. This work was published in the Journal of Animal Reproduction Science in 2011.

4. The degree of embryonic death from cold egg storage has a genetic component. Holding eggs for up to 10 days in a cold room prior to incubation is a common practice in the broiler industry that can have a negative impact on embryo survival. An ARS-Beltsville scientist investigated the impact of cold storage on the embryo survival of eggs from two different hen lines. While nearly identical before cold storage, the embryonic cell numbers, embryo diameters, and percentage of viable cells differed between the two lines after cold storage. Of interest is that 22% of the embryos from one line died after the onset of blood formation, whereas 45% of the embryos from the other line died after the onset of blood vessel formation. This suggests that the mechanism signaling the onset of blood vessel formation in these two hen lines are differentially impacted by egg storage.

Review Publications
Foye-Jackson, O., Long, J.A., Bakst, M.R., Blomberg, L., Akuffo, V.G., Silva, M.V., Guthrie, H.D., Mcmurtry, J.P. 2011. Oviductal expression of avidin, avidin-related protein-2 and progesterone receptor in turkey hens in relation to sperm storage: effects of oviduct tissue type, sperm presence, and turkey line. International Journal of Poultry Science. 90:1539-1547.

Guthrie, H.D., Welch, G.R., Theisen, D., Woods, L.C. 2011. Effects of hypothermic storage of striped bass (Morone saxatilis) sperm on intracellular calcium, reactive oxygen species formation, mitochondrial function, motility, and viability. Theriogenology. 75:951-961.

Pelaez, J., Bongalhardo, D.C., Long, J.A. 2011. Characterizing the glycocalyx of poultry spermatozoa; semen cryopreservation methods alter the carbohydrate component of rooster sperm membrane glycoconjugates. Poultry Science. 90(2):435-443.

Bakst, M.R. 2011. Role of the oviduct in maintaining sustained fertility in hens. Journal of Animal Science. 89:1323-1329.

Dalloul, R.A., Long, J.A., Zimin, A.V., Reed, K.M., Blomberg, L., Van Tassell, C.P., Schroeder, S.G., Sonstegard, T.S., Aslam, L., Beal, K., Biedler, J., Burt, D.W., Crasta, O., Crooijmans, R.P., Cooper, K., Coulombe, R.A., De, S., Delany, M.E., Dodgson, J.B., Dong, J.J., Evans, C., Flicek, P., Florea, L., Folkerts, O., Groenen, M.A., Harkins, T.T., Herrero, J., Hoffmann, S., Megens, H., Jiang, A., Jong, P., Kaiser, P., Kim, H., Kim, K., Kim, S., Langenberger, D., Lee, M., Lee, T., Mane, S., Marcais, G., Marz, M., Mcelroy, A.P., Modise, T., Nefedov, M., Notredame, C., Paton, I.R., Payne, W.S., Pertea, G., Prickett, D., Puiu, D., Qioa, D., Raineri, E., Salzberg, S.L., Schatz, M.C., Scheuring, C., Schmidt, C.J., Schroeder, S.G., Smith, E.J., Smith, J., Sonstegard, T.S., Stadler, P.F., Tafer, H., Tu, Z., Van Tassell, C.P., Vilella, A.J., Williams, K., Yorke, J.A., Zhang, L., Zhang, H., Zhang, Z., Zhang, Y. 2010. Multi-platform next-generation sequencing of the domestic turkey (Meleagris gallopavo) genome assembly and analysis. PLoS Biology. 8(9):e1000475.

Last Modified: 9/1/2014
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